We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens, as a model of tissue-specific gene expression. In FY2002 we have continued to examine the mouse small heat shock protein (shsp)/ alphaB-crystallin gene locus. A shsp/alphaB-crystallin-related gene (MKBP/HSPB2), which was derived by duplication of the shsp/alphaB-crystallin gene during evolution, is present 1 kb upstream of the mouse shsp/alphaB-crystallin gene and is transcribed in the opposite direction. The MKBP/HSPB2 gene is expressed in muscle and heart, but not in lens. We have shown by placing either firefly or Renilla luciferase reporter genes at each end of the linked promoters that the intergenic aB-crystallin enhancer affects the shsp/alphaB-crystallin promoter in an orientation-specific manner, and has surprisingly little influence on the MKBP/HSPB2 promoter. We have also shown that a sequence between the MKBP/HSPB2 gene and the enhancer is replete with GV-rich stretches that further insulate the MKBP/HSPB2 promoter from the enhancer. We have continued our collaborative project with Dr. Eviatar Nevo (University of Haifa, Israel) on the blind mole rat in FY2002. This rodent develops an eye during embryogenesis that regresses and has a degenerate lens fragment at best in the adult. Last year we showed that the alphaB-crystallin promoter/enhancer of the mouse and blind mole rat are similar but not identical. Luciferase reporter transgenes driven by either a mole rat or a mouse alphaB-crystallin promoter/enhancer are expressed similarly in the heart of transgenic mice. However, the mole rat promoter/enhancer is at least than 10 times more active than the mouse promoter/enhancer in skeletal muscle and barely active in lens in the transgenic mice. The mole rat promoter does function during early embryonic in the transgenic mouse, but turns off later in development, suggesting the existence of a developmental switch for shsp/alphaB-crystallin gene expression. These data indicate that the shsp/alphaB-crystallin promoter/enhancer has undergone adaptive changes corresponding to the subterranean evolution of the blind mole rat. We are presently attempting to identify the sequences regulating the mole rat alphaB-crystallin promoter in the transgenic mice. In FY2002 we also continued our studies on the jellyfish lens crystallins. Jellyfish have complex eyes with a cellular lens, cornea and retina. Last year we showed by cDNA and gene cloning that the jellyfish J3-crystallin is similar to vertebrate saposins. Saposins are multifunctional proteins that bridge lysosomal hydrolases to lipids and activate enzyme activity. Our data suggest a crystallin role for the multifunctional saposin protein family in the jellyfish lens, extend the gene sharing evolutionary strategy for lens crystallins to cnidarians, and indicate that the putative primordial J3-crystallin reflects both the chaperone and enzyme connections of the vertebrate crystallins. Moreover, we showed that the J3-crystallin gene is expressed in the statocyst, which is a mechanoreceptive balancing organ akin to the vertebrate ear, as well as the lens. We also showed that the jellyfish Pax protein, PaxB, is a structural hybrid of Pax6 (critical for eye development) and Pax2 (critical for eye and ear development), further implicating a commonality between the eye and the ear in evolution. PaxB has a Pax2/5/8 DNA-binding paired domain and octapeptide and a Pax6 homeodomain. In FY2002 we showed that PaxB activates the J3-crystallin promoter in transfection experiments, extending the fundamental role of Pax proteins for crystallin gene regulation throughout the animal kingdom. We also showed that it activates the Drosophila rh6 opsin promoter, a property attributed to Pax6 in other species. Moreover, we showed Pax2/5/8 propeties of PaxB including activation/inhibitory regions of the activation domain and the ability to rescue spapol, a Drosophila Pax2 eye mutant. Most remarkable, we showed that PaxB can induce ectopic eyes in Drosophila, a feature that in the past was believed to required a Pax6 paired domain. Thus, our data suggest that PaxB, rather than Pax6, might be the primoridal gene for eye evolution. Finally, we show expression of PaxB in the statocyst as well as the lens and retina of the jellyfish eye, suggesting that inner ears (mechanoreceptors) and eyes are evolutionarily related.